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Tenth Dr Yellapragada SubbaRow memorial Lecture of Indra Prastha University, New Delhi January 14, 2013 Enzymatic regulation of Lymphaocyte cell fate decisions and new therapies for human diseases By Shiv Pillai MBBS, PhD, Boston, MA, USA

Dr. Shiv Pillai is Professor of Medicine at Harvard Medical School and Professor of Health Sciences and Technology at Massachusetts General Hospital Cancer Center in Boston, Massachusetts, USA. After his basic medical education at Christian Medical College, Vellore, he obtained his PhD in Biochemistry working in the laboratory of Professor Bimal Bachhawat initially at Vellore and later at IICB Kolkatta. As graduate student, he developed novel chemical cross linking approaches to study the oligomeric structures of glycoprotein enzymes. As investigator at the Kothari Center of Gastroenterology at Kolkatta, he initiated translational studies on the immunology of amoebiasis. Moving to USA, he discovered surrogate light chains and the pre-B cell receptor while postdoctoral fellow at the laboratory of Prof. David Baltimore of MIT and Whitehead Institute. In 1988, he was appointed to the faculty at Harvard Medical School and MGH Cancer Center. He directs courses in Immunology at Harvard University and Harvard Medical School. pillai@helix.mgh.harvard.edu

The overriding goal of the work in our laboratory has been to obtain an in-depth understanding of the mechanisms involved in the development and activation of B lymphocytes in order to have insights into pathogenesis and make new approaches to the treatment of common disorders.

Different B lineages develop from distinct haematopoietic stem cells (HSCs).

B-1 B cells emerge from fetal liver derived HSCs and B-2 B cells from bone marrow derived HSCs. Common lymphoid progenitors commit to the B lineage and initiate rearrangement of immunoglobulin (Ig) heavy chain gene segments. Developing B cells, which make in-frame rearrangements of the Ig heavy chain gene, assemble the pre-B cell receptor and are selected to survive and expand.

Ig light chain genes are then rearranged to give rise to immature B cells with clonal B cell receptors on their cell surfaces that can engage specific antigens. Immature B cells that engage self-antigens in the bone marrow with high affinity are tolerized by a process called receptor editing.

B cells that are not strongly self-reactive exit the bone marrow and differentiate further in the spleen. A cell-fate decision at this site contributes to the generation of two mature B cell subsets - Marginal Zone B cells that mainly deal with blood-borne pathogens and Follicular B cells that can collaborate with T cells and re-circulate through the blood traveling from lymph node to lymph node in search of antigen and specific helper T cells.

Protein antigens, typically from microbes, have both B cell epitopes (“molecular bumps” that are recognized by antigen receptors on specific B cells) and internal linear peptides (T cell epitopes) that facilitate T-B collaboration during an immune response. T cell help results in the formation of germinal centers � sites at which events such as isotype switching and somatic hypermutation occur followed by selection of high affinity memory B cells and long-lived plasma cells.

B lymphocytes have functions that go beyond the production of high affinity antibodies that mediate protection against pathogens. They play important roles in the activation of specific helper T cells and in the maintenance of memory CD4+ T cells.

The depletion of B cells using anti-CD20 (Rituxan) contributes to clinical improvement in a range of diseases many of which are believed to be actually caused by T cells.

Our discovery of surrogate Ig light chains and the pre-B cell receptor (the first ever pre-antigen receptor described) predates by many years the discovery of its homolog in T cells. We also demonstrated that the pre-B cell receptor signals constitutively in a ligand independent manner � not sensing the environment but monitoring the reading frame. This model is widely accepted as the mechanism for signaling by both the pre-B cell and the pre-T cell receptors.

We also discovered that BTK (Bruton’s Tyrosine Kinase) is activated downstream of the pre-B cell receptor and the B cell receptor. BTK inhibitors have been developed by a number of companies and have recently shown tremendous promise in both lymphoid malignancies and autoimmunity.

We have also defined a novel “follicular vs. marginal zone B cell” fate decision in the spleen: different strengths of BCR/BTK signaling give rise to follicular B cells or marginal zone B cells.

Our demonstration that canonical NFkB signaling is required for marginal zone B cell development was the first report of a developmental role in lymphocytes for NFkB. We have showed that NFkB and Notch-2 work synergistically to drive marginal zone B cell development.

We identified two novel stages during B cell development � a marginal zone precursor (MZP) B cell development stage and a BTK-independent Follicular B cell development stage. We have described what we call Follicular Type II cells. We have also identified a novel peri-sinusoidal niche in the bone marrow for follicular B cells.

Both B-1 B cells and Marginal Zone B cells are long-lived self-renewing cells.

Human marginal zone B cells are also known as IgM memory cells. While we have shown that Notch 2 and NFkB are required for MZ B cell development, we asked if de novo DNA methylation is relevant to the self-renewal of long-lived B cell subsets and B cell memory, especially since memory lymphocytes display global alterations in DNA methylation. We conditionally deleted Dnmt3a in the B lineage.

Young mice displayed a marked increase of B-1 B cells in their natural habitat � the peritoneum -- although initial production of B-1 B cells was not increased. The absence of Dnmt3a leads to enhanced self-renewal of B-1 B cells. All mutant mice acquire by the third month the features of a human monoclonal B lymphocytosis, considered to be a precursor state of chronic lymphocytic leukemia (CLL). In the Dnmt3a conditional knockout mice, these expanded B-1 B cells spill by the fifth month into the blood and spleen mimicking a full-blown CLL. The leukemic cells closely phenocopy the more severe version of human CLL in which Ig genes are largely unmutated.

We will continue to conduct studies on the evolution of CLL in this remarkable mouse model looking at global methylation patterns and using Next Gen Sequencing approaches to examine changes in B cell repertoires from polyclonal stages to the clonal leukemia stage. BCR signaling is important in the disease � in human CLL BTK inhibitors result in clinical improvement in 90% of CLL subjects.
We have determined that an enzyme, sialic acid acetyl esterase (SIAE), counters the BTK pathway and is required for the maintenance of peripheral B cell tolerance and the prevention of autoimmunity. SIAE regulates inhibitory signaling in B cells by receptors of the Siglec family. This enzyme removes acetyl moieties from the 9-OH position of sialic acid and makes ligands available for inhibitory receptors of the Siglec family.

In the B lineage, the absence of this enzyme results in dampening of inhibitory signals from Siglec2/CD22 and therefore exaggerates activation of the BCR/BTK pathway. Hyperactivation of B cells results in a break in peripheral tolerance and consequent autoimmunity.

A publication in late 2012 from our laboratory follows up our earlier human studies and clarifies the relevance of this pathway in human autoimmunity.

Why do B cell have inhibitory signaling molecules like Aiolos, CD22, and SIAE? We have shown that these inhibitory pathways exist to “squelch” weakly self-reactive B cells and prevent them from getting unwanted T cell help. Such T cell help could drive these B cells to induce a cytidine deaminase called AID, undergo somatic hypermutation and become strongly autoreactive.

SIAE and Siglecs prevent promiscuous interactions between T and B cells.
From a mechanistic standpoint, phenotypic alterations that occur in mice lacking SIAE are of biological interest in their own right. We are currently exploiting these alterations in seeking novel approaches to both preventive and therapeutic immunization.

One phenomenon is a dramatic enhancement of somatic hypermutation. Increasing AID levels beyond those seen in activated B cells does not increase somatic mutation.

However, taking away SIAE causes repeated T-B interactions, sustains high AID levels and markedly enhances somatic hypermutation. We are attempting to see if this finding can be exploited to increase somatic mutation during preventive immunization against weak but conserved epitopes of immunogens like gp140 of HIV and our early results are very promising.

In conclusion, many of our fundamental discoveries about cell fate decisions and BTK signaling have already been translated into therapies in subjects with autoimmunity and with B cell malignancies.
Pharmaceutical companies have developed novel BTK inhibitors that have proven extremely successful in trials in patients with lymphoid malignancies.

After completion of clinical trials, five BTK inhibitors including Ibrutinib are now on the market.������
Our current discoveries on the role of the regulation of de novo DNA methylation in B lymphoid self-renewal are on the verge of providing novel insights into CLL pathogenesis and are likely to lead to novel therapies for CLL beyond BTK inhibitors.

Our new insights into the role of SIAE in regulating T lymphocyte-B lymphocyte collaboration may well catalyze novel approaches to enhance somatic hypermutation (for preventive vaccination) as well as T cell memory (for therapeutic vaccines).

Novel insights obtained into the role of Siglecs and SIAE in controlling lymphoid versus myeloid differentiation may well translate into newer therapies in childhood leukemias.� (Based on Professor Pillai’s summary of his presentation and his e-mailed clarification)